Structural and enzymatic studies of Organic Hydroperoxide Resistance Protein
Cussiol, J.R.R.a; Oliveira, M.A.a; Guimarães, B.G.b; Medrano, F.J.b and Netto, L.E.S.a
a From the Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo; b From Centro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Brazil.
Ohr (Organic Hydroperoxide Resistance Protein) belongs to a family of proteins that are only present in bacteria, most of wich are pathogenic to plants and animals. These proteins were characterized as thiol-dependent peroxidases where reactive cysteine residues are able to reduce peroxides. Ohr is capable to reduce organic peroxides one hundred fold more eficient than H2O2. Towards NADH consumption assay using the dithiol lipoamide (modified from Bryk et. al, 2004) we calculate kinetic parameters from Ohr in the presence of tert-butyl hydroperoxide (t-BOOH) and cumene hydroperoxide. Both peroxides presents similar results about the catalytic eficience (kcat/Km) of Ohr being calculate about 105 M-1 X s-1. Results obtained during the last refinement cycles from Ohr structure showed an elongated electron density in the entrance of active site pockets corresponding to a polyethylene glicol (PEG) molecule derived from the crystallization solution. PEG molecule interacts with several residues in the active site asnd presented several hydrofobic contacts. Crystal contacts from two different structures indicate that the PEG binding is not a mere artifact, but it may mimic the binding of some endogenous substrates. Molecular modeling studies showed that peroxides derived from oleic acid perfectly accomodates in the active site. Finally, we showed that only PEG 400 molecule is capable to inhibit Ohr peroxidase activity. We test several another PEG molecules from different molecular weights but none of them were capable to inhibit Ohr activity.
|